Hi,

I am (at this Moment) also cloning px458 and px459 (both based on px330) with 2 sgRNAs. How did you manage this?

I amplified the hU6-promoter-gRNA cassette with XbaI- and NheI-sites to ligate into XbaI-digested plasmids, backbone and insert carrying 2 different gRNAs.

I was also planning to sequence with primers annealing upstream of the first hU6 promoter (in the pBR322 origin).

An alternative approach would be taking one of the Oligos of the second gRNA that was inserted as reverse Primer.

EDIT: Sorry, I misread px333 for px330

Still you might consider my suggestion.

Best,

Felix

Hi,

Thank you very much for your answer. So far I had designed primers annealing a bit closer to the U6 promoter but it didn't work very well, I'll try with one annealing pBR322 ori.

px333 already has 2 U6 promoters: the first one is followed by BbsI restriction sites and the second one by BsaI restriction sites, so I am doing two successive cloning,  one to insert the first gRNA using BbsI (I am trying to validate this one), the  second cloning is  to insert the 2nd gRNA  using BsaI.

Here is a link http://blog.addgene.org/c rispr-101-multiplex-expression -of-grnas

Best,

Awa

Hi,

I had this problem while working with other vector, there may be sequence duplication in the plasmid. My suggestion is to check annealing site of your sequencing primer by using software (link). Good luck.

http://www.bioinformatics.org/seqext/

Hello,

I am working with pX333 and used the forward sequencing primer" TTG CTC ACA TGC GCC ATT CT", sent it out to genescript per their protocol and it came back perfect. The primer binds just upstream of the first U6 promoter. Hope this helps!

Hi,

Many thanks for your answers, I managed to do it using a primer annealing the pBR322 ori. The double knockdown worked well!

Hello

I would like to insert two guide RNA into PX333, Can you give me please the protocol that you used?

Heba